Outbreaks of Escherichia coli O157:H7 Infections Linked to Romaine Lettuce in Canada from 2008 to 2018: An Analysis of Food Safety Context.

ABSTRACT: Foodborne diseases are a major cause of illness in Canada. One of the main pathogens causing cases and outbreaks of foodborne illness in Canada is Escherichia coli O157:H7. From 2008 to 2018, 11 outbreaks of E. coli O157:H7 infection in Canada were linked to leafy greens, including 7 (63.6%) linked to romaine lettuce, 2 (18.2%) linked to iceberg lettuce, and 2 (18.2%) linked to other or unspecified types of leafy greens. The consumption of lettuce in Canada, the behavior of E. coli O157:H7 on lettuce leaves, and the production practices used for romaine and iceberg lettuce do not seem to explain why a higher number of outbreaks of E. coli O157:H7 infection were linked to romaine than to iceberg lettuce. However, the difference in the shape of iceberg and romaine lettuce heads could be an important factor. Among the seven outbreaks linked to romaine lettuce in Canada between 2008 and 2018, an eastern distribution of cases was observed. Cases from western provinces were reported only twice. The consumption of romaine and iceberg lettuce by the Canadian population does not seem to explain the eastern distribution of cases observed, but the commercial distribution, travel distances, and the storage practices used for lettuce may be important factors. In the past 10 years, the majority of the outbreaks of E. coli O157:H7 infection linked to romaine lettuce occurred during the spring (March to June) and fall (September to December). The timing of these outbreaks may be explained by the availability of lettuce in Canada, the growing region transition periods in the United States, and the seasonality in the prevalence of E. coli O157:H7. The consumption of romaine lettuce by the Canadian population does not explain the timing of the outbreaks observed.

Outbreak of Vibrio parahaemolyticus Associated with Consumption of Raw Oysters in Canada, 2015.

There has been a steady increase in illness incidence of Vibrio parahaemolyticus (Vp). The majority of illnesses are associated with consumption of raw oysters. In the summer of 2015, Canada experienced the largest outbreak associated with the consumption of raw oysters harvested from British Columbia (BC) coastal waters. Case investigation of laboratory-confirmed cases was conducted to collect information on exposures and to assist traceback. Investigations at processors and oyster sampling were conducted. Eighty-two laboratory-confirmed cases of Vp infection were reported between January 1 and October 26, 2015. The majority of the cases were reported in BC, associated with consumption of raw BC oysters in restaurants. Sea surface temperatures were above the historical levels in 2015. This outbreak identified the need to improve surveillance and response to increases in human cases of Vp. This is of particular importance due to the potential for increasing water temperatures and the likelihood of additional outbreaks of Vibrio.

Development of a method for the detection of verotoxin-producing Escherichia coli in food.

The growing recognition of the role of non-O157 verotoxigenic Escherichia coli (VTEC) in foodborne illness underscores the importance of developing methods to detect it in the food supply. We describe here the development of a protocol for the detection, isolation, and characterization of VTEC from foods, designed for the serotype-independent enrichment, detection, and isolation of VTEC, in combination with rapid characterization of VTEC O157, O26, O103, O111, and O145. This study examined the inhibitory concentration of six antimicrobial agents used either singly or in combination for the optimal enrichment of a panel of 18 different O serogroups of VTEC in modified tryptic soy broth. Considerable variability in resistance to the different antimicrobials tested was noted among different VTEC strains. The combination enabling growth of strains of all 18 different O serogroups was vancomycin (10 µg/ml) and cefsulodin (3 µg/ml). A similar combination of antimicrobials formulated in agar plates was found beneficial in the recovery of VTEC strains from enrichment broth cultures. The efficacy of these media in the recovery of selected VTEC (O26, O103, O111, O145, and O157) from ground beef and O157 VTEC from lettuce, spinach, and apple cider was demonstrated. The selective enrichment media described herein would appear suitable for incorporation in methods for the recovery and detection of a wide range of VTEC serogroups.

A simple PCR-based macroarray system for detection of multiple gene markers in the identification of priority enterohemorrhagic Escherichia coli.

Enterohemorrhagic Escherichia coli (EHEC) strains bearing the O antigenic determinants O157, O26, O111, O103, and O145 have a high rate of association with foodborne illness worldwide. To expand Canadian food inspection capability, a cloth-based hybridization array system (CHAS) was developed for the identification and characterization of priority EHEC. This method targets key virulence genes (eae, hlyA, vt1, and vt2) plus the rfbE gene specifying the O157 antigenic determinant, and the wzx genes specifying the O26, O111, O103, and O145 determinants. Multiplex PCR products incorporating a digoxigenin label were detected by hybridization with an array of specific oligonucleotide probes immobilized on a polyester cloth support, with subsequent immunoenzymatic assay of the captured amplicons. This method identified the relevant markers in 85 different strains bearing various combinations of the target genes (virulence and priority O-antigen markers). None of the target genes was detected in 26 different strains of other E. coli and non-E. coli bacteria. The CHAS demonstrated 100% inclusivity and 100% exclusivity characteristics, with respect to detection of the various markers among different bacterial strains. The CHAS demonstrated 100% inclusivity and 100% exclusivity characteristics, with respect to detection of the markers among various target and nontarget bacteria. The entire procedure could be completed in less than 5 h, and is useful for the identification of priority EHEC colonies isolated from foods by using enrichment culture techniques.

Development of unique bacterial strains for use as positive controls in the food microbiology testing laboratory.

Nalidixic acid-resistant (NalR) mutants of Salmonella enterica serovar Berta and Escherichia coli O157:H7 were derived from wild-type laboratory cultures to serve as distinguishable control strains for routine use in food microbiology testing programs. The prevalence of the NalR phenotype among different bacteria was verified using panels of related and unrelated strains with the ability to grow vigorously on plating media containing nalidixic acid, being restricted to the NalR mutants. The NalR phenotype was stable in both mutant strains over several generations in the absence of selective pressure and enabled their differentiation from wild-type bacteria on the basis of their ability to grow on plating media containing nalidixic acid. A similar approach for the development of a distinguishable Listeria monocytogenes control strain was not possible due to the inherent resistance of this organism to nalidixic acid. Instead, an L. monocytogenes isolate with rare genotypic and serologic features was identified as a possible candidate to serve as a unique and distinguishable positive control strain.

Rapid discrimination of Salmonella isolates by single-strand conformation polymorphism analysis.

A molecular typing technique was developed for the differentiation of Salmonella isolates based on single-strand conformation polymorphism (SSCP) analysis of amplicons generated by PCR. Amplicons from parts of the fimA (both the 5' and 3' ends), mdh, invA, and atpD genes were generated separately from a panel of Salmonella strains representing Salmonella bongori, and four subspecies and 17 serovars of Salmonella enterica. These amplicons were subjected to SSCP analysis for differentiation of the salmonellae on the basis of different conformational forms arising due to nucleotide sequence variations in the target genes. Several distinct SSCP banding patterns (a maximum of 14 each for atpD and fimA 3' end) were observed with this panel of Salmonella strains for amplicons generated from each target gene. The best discrimination of Salmonella subspecies and serovar was achieved from the SSCP analysis of a combination of at least three gene targets: atpD, invA, and either mdh or fimA 3' end. This demonstrates the applicability of SSCP analysis as an important additional method to classical typing approaches for the differentiation of foodborne Salmonella isolates. SSCP is simple to perform and should be readily transferable to food microbiology laboratories with basic PCR capability.

Detection of group D salmonellae including Salmonella Enteritidis in eggs by polymyxin-based enzyme-linked immunosorbent assay.

A high-throughput, rapid method was devised for the detection of Salmonella Enteritidis in egg products. For each target organism, preenrichment in nutrient broth was followed by selective enrichment in Rappaport-Vassiliadis soya peptone and tetrathionate brilliant green broths or by plating on modified semisolid Rappaport Vassiliadis (MSRV) agar medium. The presence of Salmonella Enteritidis was determined by subjecting portions of the selective broth cultures or swarming growth on MSRV medium to an enzyme-linked immunosorbent assay (ELISA) procedure using polymyxin immobilized in the wells of a microtiter plate as a high-affinity adsorbent for lipopolysaccharide (LPS) antigens. Sample extracts were reacted with polymyxin-coated microwells, and captured LPS antigens were detected immunoenzymatically with a commercially available Salmonella factor O9-specific antibody. The polymyxin-ELISA was 100% sensitive and 100% specific for Salmonella strains bearing the O9 antigen. When the ELISA was combined with enrichment using either the selective broths or plating on MSRV medium, the system was an effective means for detection of Salmonella Enteritidis in artificially inoculated egg products. The polymyxin-ELISA is a simple and inexpensive assay for group D salmonellae (including Salmonella Enteritidis) in a convenient 96-well microtiter plate format, making this system ideally suited for processing large numbers of samples.

Polymyxin-based enzyme-linked immunosorbent assay for the detection of Escherichia coli O111 and O26.

Polymyxin-based enzyme-linked immunosorbent assay (polymyxin-ELISA) systems were developed for the detection of Escherichia coli O111 and O26 in ground beef after enrichment. Polymyxin immobilized in the wells of a microtiter plate served as a high affinity adsorbent for lipopolysaccharide (LPS) antigens, which were detected immunoenzymatically using commercially available anti-E. coli O111 or anti-E. coli O26 antisera. The polymyxin-ELISA sensitively detected E. coli strains bearing the O111 and O26 LPS antigens, discriminating between these target strains and a panel of various non-target Gram negative and Gram positive bacteria. The detection of E. coli O111 and O26 strains inoculated into ground beef was achieved after enrichment in either modified trypticase soy broth (TSB) with novobiocin, or the serotype-specific medium TSB supplemented with cefixime and vancomycin (E. coli O111), and the same medium containing potassium tellurite (E. coli O26). The polymyxin-ELISA shows promise as a rapid, simple and inexpensive screening tool for E. coli O111 and O26 in enrichment cultures of ground beef.

Plasmid vectors for protein production, gene expression and molecular manipulations in Aspergillus niger.

We constructed three sets of plasmids for use in Aspergillus niger. These plasmids were assembled using various combinations of a series of modular DNA cassettes that included a selectable marker, pyrG, derived from Aspergillus nidulans; two promoter regions for directing protein expression; a cassette derived from the AMA1 replicator sequence to support autonomous replication; and a reporter gene based on the A. niger lacA gene. One set included integrating and autonomously replicating plasmids for the expression of homologous and heterologous proteins. The second was a set of autonomously replicating plasmids, with a secreted beta-galactosidase encoding reporter gene, for studying gene regulation events. The third set included pyrG-derived gene-blaster cassettes suitable for genome manipulation by targeted gene replacement.

Detection of Escherichia coli O157 in foods by a novel polymyxin-based enzyme-linked immunosorbent assay.

An enzyme-linked immunosorbent assay (ELISA) system was developed using polymyxin immobilized in the wells of a microtiter plate as a high-affinity adsorbent for Escherichia coli O157 lipopolysaccharide (LPS) antigens. Extracts from cell suspensions were reacted with polymyxin-coated microwells followed by immunoenzymatic detection of captured LPS antigens using a commercially available anti-E. coli O157 antibody-peroxidase conjugate and a 3,3',5,5'-tetramethylbenzidine substrate. The polymyxin ELISA was highly sensitive and specific for E. coli strains bearing the O157 antigen. When this ELISA was combined with enrichment, results were in complete agreement with those of standard culture techniques for the detection of this pathogen in a variety of artificially inoculated and naturally contaminated foods. The polymyxin ELISA is a simple and inexpensive assay for E. coli O157 with a 96-well microtiter plate format, making this system ideally suited for processing large numbers of samples.